Thirty percent of patients with severe hemophilia treated with factor fVIII (fVlll) develop anti-fVIII antibodies (inhibitors). This is the most significant complication in the management of patients with hemophilia A leading to significant increases In morbidity as well as cost of treatment. Antibodies to fVIII can also develop in patients with previously normal coagulation leading to acquired hemophilia. These antibodies that develop can inhibit the biological activity of fVIII and necessitate the use of bypassing agents for the treatment of bleeding episodes. However, for unknown reasons, some patients display poor hemostatic response to bypass therapy. Thus, improved treatment options are needed, especially for severe or life-threatening bleeding events. Some biological determinants have been identified that increase the risk of inhibitor development. However, little is known about how hemophilia genotype relates to the ultimate epitope spectrum of the B-cell response or how the epitope spectrum affects the response to treatment and immune tolerance. We have recently shown that epitope specificity and inhibitor kinetics within the C2 domain are more important than inhibitor titer in response to fVlll in acquired hemophilia plasma and a murine monoclonal antibody (MAb) system. The central hypothesis of this project is that the inhibitor epitope spectrum of an individual patient can be used to predict the response to infusions of fVIII with or without bypassing agents. We propose to define further the epitopes on fVIII using a large panel of non-overlapping antl-fVIII MAbs in multiple in vitro assays (Aim 1). Given known discrepancies between the different in vitro assays and bleeding phenotype we will study the importance of different epitopes on murine in vivo bleeding phenotypes and fibrin clot structure (Aim 2). In Aim 3, we will adapt our novel ELISA-based epitope mapping protocol to a more robust microsphere-based assay and investigate in a cross-sectional study how polyclonal patient plasma epitope maps correlate with the response to fVIII and bypassing agents in vitro. Ultimately these results could provide a simple clinical test that may predict response to fVIII and bypassing agents in high titer acquired and congenital hemophilia patients.